LPS, but not GM-CSF or DEX, led to NF-κB–binding activity. (A) PMNs were either left untreated or treated with 1 μg/mL LPS, 20 ng/mL GM-CSF (CSF), or 10–7 M DEX for 60 minutes; nuclear extracts were prepared and analyzed by EMSA using an H2K-binding site probe for NF-κB (n = 3). Only LPS increased NF-κB–binding activity in PMNs. The amount of nuclear extract used for the binding reaction was 5 μg protein for all samples. (B) Specificity of the bands was assessed by competition with a cold probe versus a mutated cold probe and supershift experiments. A 20-fold excess of unlabeled NF-κB probe and mutated cold probe and 5 specific antibodies against the family of the different members of the NF-κB family and one irrelevant antibody were added to nuclear extracts before incubation with labeled NF-κB oligonucleotide probe (n = 3). Two specific bands could be identified: the lower band as the p50/p50 homodimer and the upper band as the p50/p65 heterodimer.
